Structure-Based Characterization and Improvement of an Enzymatic Activity of Acremonium alcalophilum Feruloyl Esterase

Bacteria and fungi utilize carbohydrate-active enzymes, such as feruloyl esterases (FAEs), to degrade lignocellulosic biomass. FAEs in subfamily 5 (SF5) of carbohydrate esterase family 1 target larger substrates, making them particularly interesting. However, their mechanisms are not well understood due to limited structural information. This study presents the first structure of the catalytic domain (CD) of an SF5 FAE from Acremonium alcalophilum (AaFaeD), both free and in a complex with ferulic acid (FA). FA binds within a hydrophobic cleft formed by two hydrophobic walls facing each other. Structure-based functional mutagenesis of key residues in these walls clarified their roles in catalysis. Notably, the F120Y mutant of the AaFaeD catalytic domain (AaFaeD-CD) showed a 1.5-fold increase in catalytic activity toward methyl ferulate compared with the wild type. Structural comparisons with SF2 and SF3 FAEs revealed a more open substrate-binding site in SF5. High-performance liquid chromatography and gas chromatography-mass spectrometry analysis of destarched wheat bran hydrolysis by AaFaeD-CD showed that SF5 FAEs can process both monomeric and dimeric phenolic substrates, like 5,5 '-dehydrodiferulate, unlike SF2 and SF3 FAEs, which prefer monomeric substrates.